An absolute requirement for HIV-1 infection is expression of CCR5 on the cell surface of specific leukocyte subsets. Delineating the molecular mechanisms that control the expression of CCR5 is of importance to our understandingHIV-1 pathogenesis, and this fact along with our quest to elucidate how genetic variation in the CCR5 gene influence its transcriptional/epigenetic machinery, and eventually its expression and coreceptor efficiency is the major focus of our research program and of this competing renewal. Our research will be guided by the hypotheses indicated in the following 2 specific aims: Specific Aim 1 will test the overall hypothesis that in primaryHIV-1 target cells the constitutive, inducible and cell-type specific expression of CCR5 is regulated by the intricate interplay of two promoters. There is currently only limited knowledge of the regulation of CCR5in primary cells, and this information is critical to understanding the in vivo determinants influencing CCR5 density on the cell surface. Thus, we propose to use novel cellular systems in conjunction with innovative primary cell transfection strategies, "phylogenetic footprinting" ,DNA-protein pull-down assays coupled to high-through put array based profiling of transcription factors (TFs), RNAi together with HIV-derived vector mediated transactivation techniques to manipulate primary cell TF levels, and ChIP assays of in vivo occupancy of TFs in order to elucidate the genetic machinery that regulatesCCR5gene expression in primary cells. We are well positioned to extend these studies to address the in vivo biological relevance of specific CCR5 activating or repressing TFs by determining their role in disease course. Specific Aim 2 will test the overall hypothesis that CCR5 polymorphisms mediate their HIV-phenotypic effects by influencing combinatorial transcriptional control and epigenetic events that collectively nfluenceCCR5surface expression and coreceptor activity. Given the increasing importance of epigenetics in controlling gene expression, in this aim we will use ChIP assays, DNase I hypersensitivity and methylation assays to test the novel hypothesis that mutations in the CCR5 promotermediatetheir effects by influencing epigenetic events such as chromatin remodeling, DNA accessibility, and methylation. We will also extend significantly our ongoing studies aimed at understanding the mechanisms by which CCR5 polymorphisms disrupt combinatorial transcriptional control in a cell-type specific manner. These in vitro studies will be eventually extended to evaluate the association between the altered transcriptional/epigenetic events to established clinical genotype-HIV phenotype relationships as well as CCR5 surface levels/coreceptor efficiency. The proposed research is significant because it will provide critical insights into the molecular mechanisms that regulate the constitutive, inducible and cell-type specific expression of CCR5, and elucidate the complex interplay between HIV infection/disease-modifying polymorphisms in CCR5 and their impact on gene expression in primary HIV-target cells. By bridging this knowledge gap we will have a better understanding of the CCR5- (e.g. SNPs) as well as non-CCR5-dependent mechanisms underlying variable susceptibility to HIV (e.g. variable levels of critical CCR5-regulatingTFs that in turn impact on CCR5 levels).